Project Summary The objective of this study is to investigate and develop long-term storage of cells using cryopreservation in the context of improving health of islets for islet transplantation as a Type1 Diabetes treatment. The goal is to develop a high subzero cryopreservation method that cools cells inside protective immunoisolation devices to extend preservation time to weeks and months. Our approach is based on the best strategies employed by freeze-tolerant and hibernating animals in nature augmented with complementary strategies developed using recent scientific understanding and bioengineering principles. Importantly, our approach does not seek to solve all the problems needed for vitrification or classical cryopreservation, but rather be the first to develop tissue preservation in a controlled, partially frozen equilibrium state using high subzero temperatures (ranging from -5 to -20 C) combined with metabolic depression. These are temperatures and strategies used in nature by species able to survive months in a state of ?suspended animation,? with the whole animal, including every single organ being ?banked? without injury. Specific Aim 1 (SA1) is to select and optimize the cryostasis cocktail with components that are essential for cell survival post freezing. Non-metabolizable 3-O-methyl-d- glucose (3-OMG) and trehalose mix will be used to prevent cellular dehydration and intracellular ice formation during cryopreservation. Unisol will be used as the base solution for testing the interaction of multiple doses of 3-OMG, trehalose with storage time as a function of temperature using ?TC3 cells. Specific Aim 2 (SA2) is to achieve active suppression of metabolic rate and enhancement of stress tolerance. ?TC3 cells will be stored in the cryostasis cocktail developed in SA1 as well with multiple dose testing of antioxidants (AO), anti-apoptotic (AA) and metabolic suppressors (MS). Human islets will be cryopreserved and stored in the standard culture, in DMSO, and in the optimal dosage, additives, temperature and storage duration established through SA1 and SA2. Quality assessments of these islets include oxygen consumption rate (OCR) normalized to DNA, percent recovery as measured by DNA to assess viability, and glucose stimulated insulin secretion to measure function. Specific Aim 3 (SA3) is to test encapsulated islets in the TheraCyte device using the most promising cryostasis cocktail established from SA1 and SA2. Islets will be cryopreserved and stored using the standard culture methods and the best conditions from SA1 and SA2. Once thawed, islet quality assessments from SA2 will be performed immediately post-thaw and after the 48 hour culture recovery period. Specific aim 4 (SA4) is to test cryopreserved, encapsulated human islets in a diabetic nude mouse model. Human islets within Theracyte immunoisolation devices that have been cryopreserved and stored in standard culture and the new cryostasis cocktail will be transplanted into diabetic nude mice. Mice will be monitored for diabetes reversal by taking daily blood glucose and weekly intraperitoneal glucose tolerance tests over 30 days. Explanted devices will be assessed by device OCR/DNA, GSIS, and histology to analyze islet health within the device.